Forward genetic screening in the zebrafish affords an unparalleled opportunity to discover previously elusive tumor suppressor genes whose alteration leads to disruption of the developing peripheral sympathetic nervous system (PSNS) and ultimately to overt neuroblastoma (NB), a biologically enigmatic, often fatal tumor in young children. This proposal tests two linked hypotheses: (i) genome-wide ethylnitrosourea (ENU) mutagenesis screens in the zebrafish can be used to identify dominant and recessive mutations that cause a deficiency or abnormal distribution of the PSNS, implicating genes important in NB pathogenesis and normal PSNS development; and (ii) transgenic zebrafish overexpressing the human MYCN oncogene in the developing PSNS can be used to determine the role of MYCN amplification in aberrant sympathetic neuroblast proliferation, survival and differentiation. These transgenic fish can also be mated to fish carrying candidate NB tumor suppressor genes identified in the ENU screen, to define the genetic pathways that cooperate with MYCN overexpression to induce early onset neuroblastoma. The zebrafish tyrosine hydroxylase (zTH) gene has already been cloned for use as a PSNS developmental marker in these screens, and its specificity for cells of the PSNS lineage was demonstrated by RNA in situ analysis. In Aim 1, mutant fish identified by in situ hybridization following ENU mutagenesis will be analyzed to determine the developmental stage at which the mutation occurred (neural crest vs. sympathetic neuroblast development). Next, the chromosomal location of each mutation will be mapped on the zebrafish genome, and examined for synteny with known regions of loss of heterozygosity (LOH) and deletion in human NB samples. Positional cloning will focus on genes most likely to have deleted or mutated counterparts in human NB. In Aim 2, transgenic zebrafish lines will be generated by using the zebrafish tyrosine hydroxylase (TH) promoter to drive expression of human MYCN in sympathetic neuroblasts. These lines will then be analyzed for their predisposition toward spontaneous tumorigenesis and crossed to the mutants isolated in Aim 1, to identify the combinations of genetic lesions leading to accelerated NB formation. Mutant zebrafish lines that harbor mutations in homologues of human tumor suppressor genes should provide reliable animal models for elucidating the molecular pathways leading to NB. A long-range goal is to use these models as a starting point for second-generation modifier screens to identify suppressors and enhancers of the genes causing PSNS defects, which may then be exploited as targets for therapeutic interventions in human NB.